Bispecific antibodies

Abstract

Various bispecific antibodies that specifically bind to both blood coagulation factor IX/activated blood coagulation factor IX and blood coagulation factor X and functionally substitute for the cofactor function of blood coagulation factor VIII, that is, the function to promote activation of blood coagulation factor X by activated blood coagulation factor IX, were produced. From these antibodies, multispecific antigen-binding molecules having a high activity of functionally substituting for blood coagulation factor VIII were successfully discovered.

The invention claimed is: 
     
       1. A bispecific antibody that specifically binds to blood coagulation factor IX and/or activated blood coagulation factor IX, and specifically binds to blood coagulation factor X, wherein the bispecific antibody comprises:
 a first antibody H chain comprising a variable region comprising complementarity determining regions (CDRs) 1, 2, and 3 that comprise SEQ ID NOs: 105-107, respectively; 
 a second antibody H chain comprising a variable region comprising CDRs 1, 2, and 3 that comprise SEQ ID NOs: 126-128, respectively; and 
 identical first and second antibody L chains, each L chain comprising a variable region comprising CDRs 1, 2, and 3 that comprise SEQ ID NOs: 156-158, respectively. 
 
     
     
       2. A pharmaceutical composition comprising the bispecific antibody of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
       3. A kit comprising the bispecific antibody of  claim 1 , packaged with instructions for use. 
     
     
       4. A bispecific antibody produced by a method comprising culturing a host cell or host cells comprising nucleic acid or nucleic acids encoding a bispecific antibody, wherein the bispecific antibody comprises:
 a first antibody H chain comprising a variable region comprising CDRs 1, 2, and 3 that comprise SEQ ID NOs: 105-107, respectively; 
 a second antibody H chain comprising a variable region comprising CDRs 1, 2, and 3 that comprise SEQ ID NOs: 126-128, respectively; and 
 identical first and second antibody L chains, each L chain comprising a variable region comprising CDRs 1, 2, and 3 that comprise SEQ ID NOs: 156-158, respectively. 
 
     
     
       5. The bispecific antibody of  claim 4 , wherein the method further comprises obtaining the bispecific antibody produced by the cultured host cell or host cells. 
     
     
       6. A pharmaceutical composition comprising the bispecific antibody of  claim 5  and a pharmaceutically acceptable carrier. 
     
     
       7. A kit comprising the bispecific antibody of  claim 5 , packaged with instructions for use. 
     
     
       8. The bispecific antibody of  claim 1 , wherein:
 CDRs 1, 2, and 3 of the first antibody H chain consist of SEQ ID NOs: 105-107, respectively; 
 CDRs 1, 2, and 3 of the second antibody H chain consist of SEQ ID NOs: 126-128, respectively; and 
 CDRs 1, 2, and 3 of each identical L chain consist of SEQ ID NOs: 156-158, respectively. 
 
     
     
       9. The bispecific antibody of  claim 1 , wherein the first antibody H chain variable region comprises SEQ ID NO: 45. 
     
     
       10. The bispecific antibody of  claim 1 , wherein the second antibody H chain variable region comprises SEQ ID NO: 52. 
     
     
       11. The bispecific antibody of  claim 1 , wherein the identical first and second antibody L chain variable regions each comprise SEQ ID NO: 62. 
     
     
       12. The bispecific antibody of  claim 1 , wherein the first antibody H chain consists of SEQ ID NO: 20. 
     
     
       13. The bispecific antibody of  claim 1 , wherein the second antibody H chain consists of SEQ ID NO: 25. 
     
     
       14. The bispecific antibody of  claim 1 , wherein the identical first and second antibody L chains each consist of SEQ ID NO: 32. 
     
     
       15. A bispecific antibody produced by a method comprising culturing a host cell or host cells comprising nucleic acid or nucleic acids encoding a bispecific antibody, wherein the bispecific antibody comprises:
 a first antibody H chain comprising a variable region comprising CDRs 1, 2, and 3 that consist of SEQ ID NOs: 105-107, respectively; 
 a second antibody H chain comprising a variable region comprising CDRs 1, 2, and 3 that consist of SEQ ID NOs: 126-128, respectively; and 
 identical first and second antibody L chains, each L chain comprising a variable region comprising CDRs 1, 2, and 3 that consist of SEQ ID NOs: 156-158, respectively. 
 
     
     
       16. The bispecific antibody of  claim 15 , wherein the method further comprises obtaining the bispecific antibody produced by the cultured host cell or host cells. 
     
     
       17. A pharmaceutical composition comprising the bispecific antibody of  claim 16  and a pharmaceutically acceptable carrier. 
     
     
       18. A kit comprising the bispecific antibody of  claim 16 , packaged with instructions for use. 
     
     
       19. A bispecific antibody that specifically binds to blood coagulation factor IX and/or activated blood coagulation factor IX, and specifically binds to blood coagulation factor X, wherein the bispecific antibody comprises:
 a first antibody H chain comprising a variable region comprising SEQ ID NO: 45; 
 a second antibody H chain comprising a variable region comprising SEQ ID NO: 52; and 
 identical first and second antibody L chains, each comprising a variable region comprising SEQ ID NO: 62. 
 
     
     
       20. A bispecific antibody produced by a method comprising culturing a host cell or host cells comprising a nucleic acid or nucleic acids encoding a bispecific antibody, wherein the bispecific antibody comprises:
 a first antibody H chain comprising a variable region comprising SEQ ID NO: 45; 
 a second antibody H chain comprising a variable region comprising SEQ ID NO: 52; and 
 identical first and second antibody L chains, each comprising a variable region comprising SEQ ID NO: 62. 
 
     
     
       21. The bispecific antibody of  claim 20 , wherein the method further comprises obtaining the bispecific antibody produced by the cultured host cell or host cells. 
     
     
       22. A pharmaceutical composition comprising the bispecific antibody of  claim 21  and a pharmaceutically acceptable carrier. 
     
     
       23. A kit comprising the bispecific antibody of  claim 21 , packaged with instructions for use. 
     
     
       24. The bispecific antibody of  claim 19 , wherein:
 the variable region of the first antibody H chain consists of SEQ ID NO: 45; 
 the variable region of the second antibody H chain consists of SEQ ID NO: 52; and 
 the variable regions of the identical first and second antibody L chains each consist of SEQ ID NO: 62. 
 
     
     
       25. A bispecific antibody that specifically binds to blood coagulation factor IX and/or activated blood coagulation factor IX, and specifically binds to blood coagulation factor X, wherein the bispecific antibody comprises:
 a first antibody H chain comprising SEQ ID NO: 20; 
 a second antibody H chain comprising SEQ ID NO: 25; and 
 identical first and second antibody L chains, each comprising SEQ ID NO: 32. 
 
     
     
       26. The bispecific antibody of  claim 25 , wherein:
 the first antibody H chain consists of SEQ ID NO: 20; 
 the second antibody H chain consists of SEQ ID NO: 25; and 
 each of the identical first and second antibody L chains consists of SEQ ID NO: 32. 
 
     
     
       27. A bispecific antibody produced by a method comprising culturing a host cell or host cells comprising nucleic acid or nucleic acids encoding a bispecific antibody comprising:
 a first antibody H chain comprising SEQ ID NO: 20; 
 a second antibody H chain comprising SEQ ID NO: 25; and 
 identical first and second antibody L chains, each comprising SEQ ID NO: 32. 
 
     
     
       28. The bispecific antibody of  claim 27 , wherein the method further comprises obtaining the bispecific antibody produced by the cultured host cell or host cells. 
     
     
       29. A pharmaceutical composition comprising the bispecific antibody of  claim 28  and a pharmaceutically acceptable carrier. 
     
     
       30. A kit comprising the bispecific antibody of  claim 28 , packaged with instructions for use. 
     
     
       31. The bispecific antibody of  claim 1 , wherein the bispecific antibody:
 (a) has increased activated blood coagulation factor X generation-promoting activity as compared to a reference antibody that consists of: (i) an antibody H chain consisting of SEQ ID NO: 165, (ii) an antibody H chain consisting of SEQ ID NO: 166, and (iii) two antibody L chains, each consisting of SEQ ID NO: 167; or 
 (b) exhibits less inhibition of F.Xase activity as compared to the reference antibody; or 
 (c) both (a) and (b). 
 
     
     
       32. The bispecific antibody of  claim 1 , wherein, when activated blood coagulation factor X generation-promoting activity of a test sample comprising the bispecific antibody is determined in an assay in which the readout is absorbance at 405 nm, the test sample produces an absorbance readout that is more than 0.4 greater than the absorbance at 405 nm produced from an identically-assayed control sample, wherein the control sample is identical to the test sample except lacking any antigen-binding molecule, and wherein the assay comprises steps (a)-(e):
 (a) incubating for 30 minutes at room temperature a first reaction mixture comprising: either (i) 5 μL of the test sample in which 300 μg/mL of the bispecific antibody is diluted in Tris-buffered saline containing 0.1% bovine serum albumin, or (ii) 5 μL of the control sample; 2.5 μL of 27 ng/mL human factor IXa beta in Tris-buffered saline containing 0.1% bovine serum albumin, 93.75 μM synthetic phospholipid solution, 7.5 mM CaCl 2 , and 1.5 mM MgCl 2 ; and 2.5 μL of 6 IU/mL human blood coagulation factor IX in Tris-buffered saline containing 0.1% bovine serum albumin, 93.75 μM synthetic phospholipid solution, 7.5 mM CaCl 2 , and 1.5 mM MgCl 2 ; 
 (b) immediately after the incubation of (a), adding to the first reaction mixture 5 μL of 24.7 μg/mL human factor X in Tris-buffered saline containing 0.1% bovine serum albumin, 93.75 μM synthetic phospholipid solution, 7.5 mM CaCl 2 , and 1.5 mM MgCl 2 , to generate a second reaction mixture, and incubating the second reaction mixture for 10 minutes at room temperature; 
 (c) immediately after the incubation of (b), adding to the second reaction mixture 5 μL of 0.5 M ethylenediaminetetraacetic acid (EDTA) in water, to generate a third reaction mixture; 
 (d) immediately after (c), adding to the third reaction mixture 5 μL of 1.47 mg/mL N-benzoyl-L-isoleucyl-L-glutamyl-glycyl-L-arginine-p-nitroaniline hydrochloride in purified water, to generate a fourth reaction mixture, and incubating the fourth reaction mixture for 50 minutes at room temperature; and 
 (e) immediately after the incubation of (d), determining the absorbance of the fourth reaction mixture at 405 nm. 
 
     
     
       33. The bispecific antibody of  claim 1 , wherein, when inhibition of F.Xase activity of a test sample comprising the bispecific antibody is determined in an assay in which the readout is absorbance at 405 nm, the test sample produces an absorbance readout that is greater than the absorbance at 405 nm produced from an identically-assayed control sample, wherein the control sample is identical to the test sample except lacking any antigen-binding molecule, and wherein the assay comprises steps (a)-(f):
 (a) incubating for 30 minutes at room temperature a first reaction mixture comprising: either (i) 5 μL of the test sample in which 300 μg/mL of the bispecific antibody is diluted in Tris-buffered saline containing 0.1% bovine serum albumin, or (ii) 5 μL of the control sample; and 2.5 μL of 80.9 ng/mL human factor IXa beta in Tris-buffered saline containing 0.1% bovine serum albumin, 93.75 μM synthetic phospholipid solution, 7.5 mM CaCl 2 , and 1.5 mM MgCl 2 ; 
 (b) immediately after the incubation of (a), adding to the first reaction mixture 2.5 μL of 1.8 IU/mL F.VIIIa in Tris-buffered saline containing 0.1% bovine serum albumin, 93.75 μM synthetic phospholipid solution, 7.5 mM CaCl 2 , and 1.5 mM MgCl 2 , to generate a second reaction mixture, and incubating the second reaction mixture for 30 seconds at room temperature; 
 (c) immediately after the incubation of (b), adding to the second reaction mixture 5 μL of 24.7 μg/mL human factor X in Tris-buffered saline containing 0.1% bovine serum albumin, 93.75 μM synthetic phospholipid solution, 7.5 mM CaCl 2 , and 1.5 mM MgCl 2 , to generate a third reaction mixture, and incubating the third reaction mixture for six minutes at room temperature; 
 (d) immediately after the incubation of (c), adding to the third reaction mixture 5 μL of 0.5 M EDTA, to generate a fourth reaction mixture; 
 (e) immediately after (d), adding to the fourth reaction mixture 5 μL of 1.47 mg/mL N-benzoyl-L-isoleucyl-L-glutamyl-glycyl-L-arginine-p-nitroaniline hydrochloride in purified water, to generate a fifth reaction mixture, and incubating the fifth reaction mixture at room temperature for 14 minutes; and 
 (f) immediately after the incubation of (e), determining the absorbance of the fifth reaction mixture at 405 nm.